This proposal focuses on the molecular basis for diapause (dormancy) in the northern house mosquito, Culex pipiens. In the early autumn adult females of this species seek protected sites and remain there throughout the winter months in a reproductively inactive state know as diapause. Although many aspects of the physiology and environmental control of diapause are well known, there is little information concerning the molecular basis for this diapause or the diapause of any other vector species. Obtaining such information is critical not only for understanding regulation of seasonal cycles of mosquitoes, but also for understanding the seasonality of pathogens the mosquito transmit. In the case of Cx. pipiens the overwintering females appear to harbor West Nile virus, and thus serve as the mechanism for reintroducing the virus into bird populations the following spring. Our initial studies using suppressive subtractive hybridization (SSH) have identified four categories of genes that are upregulated in early diapause. These include genes involved in the diapause-associated switch from blood feeding to sugar feeding, the arrest of development, cold-hardening, and responding to juvenile hormone. Similar SSH experiments will examine mid and late stages of diapause. Clones isolated with this technique will be further characterized by full-length sequencing, by evaluating expression patterns throughout diapause using northern blotting and real-time PCR, by examining tissue-specific expression patterns, and by evaluating gene function using RNA interference. We also plan to use SSH to search for genes associated with the programming phase of diapause (early pupal stage). Such genes are likely to be involved in the decision to either enter or avert diapause. At the conclusion of this study we expect to have identified major gene pathways involved in the regulation of diapause in Cx. pipiens, thus allowing us to begin the construction of a regulatory hierarchy for diapause in this species. We anticipate that these results will suggest vulnerable targets for disrupting diapause and may provide clues for how viral replication is halted during the winter months.